Impregnated disk method for testing antifungal susceptibility

Abstract

A method for testing the susceptibility of fungi to antifungal agents comprises inoculating a non-nutrient material, such as agar, with a fungus. A number of disks impregnated with various types and concentrations of antifungal agents and the proper nutrient medium for the type of fungus and antifungal agent tested is placed upon the non-nutrient medium inoculated with fungus. After an incubation period, the plates are read to determine the minimum inhibitory concentration of the particular antifungal agent for the particular fungus. The minimum fungicidal concentration can also be determined upon further incubation.

I claim: 
     
       1. A method for testing the susceptibility of a fungus to antifungal agents comprising providing dried disks consisting essentially of inert support material impregnated with nutrient medium which is capable of sustaining growth of said fungus and which does not inactivate or antifungal agent to be tested and at least one antifungal agent to be tested whereby each disk contains a particular known concentration of said antifungal agent, inoculating a non-nutrient support medium with the fungus to be tested, placing said dried impregnated disks on said inoculated non-nutrient support medium, incubating said disks and said inoculated non-nutrient support medium for a time and at a temperature capable of promoting growth of said fungus, and determining the absence or presence and extent of growth of said fungus after said incubation period. 
     
     
       2. A method according to claim 1 wherein said fungus to be inoculated on said non-nutrient medium is prepared by using a culture grown for about 24 to about 48 hours in yeast nitrogen base containing about 1% glucose and about 0.15% asparagine, centrifuging said culture, removing the supernatant, washing the remaining sedimented cells and adding sterile distilled water until a cloudy suspension results. 
     
     
       3. A method according to claim 1 wherein said non-nutrient support medium is sterile liquified 2% purified agar which has been allowed to harden. 
     
     
       4. A method according to claim 1 wherein said antifungal agent is selected from the group consisting of amphotericin B and nystatin. 
     
     
       5. A method according to claim 1 wherein said antifungal agent is 5-fluorocytosine. 
     
     
       6. A method according to claim 1 wherein said antifungal agent is diluted with said nutrient medium. 
     
     
       7. A method according to claim 1 wherein said nutrient medium comprises an aqueous solution comprising about 6.5 g/l yeast extract, about 1.5 g/l beef extract, about 5 g/l peptone, about 10 g/l pancreatic digest of casein, about 11 g/l of dextrose, about 3.5 g/l of sodium chloride, about 3.68 g/l of dipotassium phosphate and about 1.32 g/l of monopotassium phosphate or equivalent amounts thereof. 
     
     
       8. A method according to claim 1 including placing control disks impregnated with nutrient medium on said inoculated non-nutrient support medium. 
     
     
       9. A method according to claim 1 wherein said disks are dried at or below about 10° C. 
     
     
       10. A method according to claim 9 wherein said disks are dried by a lyophilization process. 
     
     
       11. A method according to claim 1 wherein said disks and inoculated non-nutrient support medium are incubated for about 24 to about 48 hours at a temperature of about 28° C. to about 31° C. 
     
     
       12. A method according to claim 11 wherein said disks and inoculated non-nutrient support medium are incubated at about 29° C. for 24 hours to determine the minimum inhibitory concentration of the tested antifungal agent. 
     
     
       13. A method according to claim 1 wherein said disks and said inoculated non-nutrient support medium are incubated for about 48 hours to determine the minimum fungicidal concentration of said antifungal agent. 
     
     
       14. A method according to claim 1 wherein said disks are impregnated with combinations of antifungal agents. 
     
     
       15. A disk-agar method for testing the susceptibility of a fungus to antifungal agent comprising impregnating disks consisting essentially of inert support material with nutrient medium which is capable of sustaining growth of said fungus and which does not inactivate an antifungal agent to be tested and at least one antifungal agent to be tested, whereby each disk contains a particular known concentration of said antifungal agent, drying said impregnated disks at a temperature at or below about 10° C., inoculating a non-nutrient agar with the fungus to be tested, placing said dried impregnated disks on said inoculated non-nutrient agar, incubating said disks and said inoculated non-nutrient agar for a time and at a temperature capable of promoting growth of said fungus, and determining the absence or presence and extent of growth of said fungus after said incubation period.